Highly branched, unsubstituted or low-substituted starch products, dialysis solution and plasma expander containing the same, and the use thereof

ABSTRACT

Known hydroxyethylated and -propylated starch types for use as colloid osmotic agent in peritoneal dialysis or as volume replacement composition (plasma expander) have the disadvantage that complete degradation by amylase is not possible owing to the more or less extensive substitution by hydroxyethyl or hydroxypropyl groups. As a consequence thereof, residual fragments remain in the body and are eliminated only very slowly or are stored in various organs/tissues, especially with relatively high and/or long-term dosage. These disadvantageous properties can be very substantially avoided according to the invention with a highly branched, unsubstituted or low-substituted starch product, i.e. with a starch which has a significantly higher degree of branching than amylopectin, and has the degree of α-I,6 branching of glycogen, or exceeds the latter and—if substituted—has a degree of substitution MS of only up to 0.3.

Highly branched, unsubstituted or low-substituted starch products,dialysis solution and plasma expander containing the same, and the usethereof.

The present invention relates to highly branched, unsubstituted orlow-substituted starch products which are suitable in particular for useas colloid osmotic agent in peritoneal dialysis and as volumereplacement composition (plasma expander).

The osmotic agent mainly employed to date in peritoneal dialysis hasbeen glucose. This has proved suitable in particular for short-termintermittent use (residence time about 2 to 3 hours), is non-toxic, isvery compatible with the other ingredients of the dialysis solution andis steam-sterilizable under nonalkaline conditions. In addition, glucoseis relatively low-cost. Nevertheless, glucose is not an ideal agentbecause unwanted side effects may occur during a peritoneal dialysis.Thus, necessarily nonphysiologically low pH values and hyperosmolarsolutions lead to irritation. Because reabsorption into the blood israpid, high blood glucose and blood lipid levels are set up. Theultrafiltration efficiency can therefore be maintained only overrelatively short periods.

Apart from avoiding side effects and excessive stress on the peritonealmembrane (the risk of peritonitis increases when the dialysis solutionis frequently changed), it was indicated particularly in continuousambulatory peritoneal dialysis (also called CAPD hereinafter) to replaceglucose by an agent which permits a longer residence time of thedialysis solution in the peritoneal cavity and thus reduces the stresson the patient. In this connection, use was made of the realization thata sufficient ultrafiltration efficiency and depletion (“clearance”) ofsolutions is achieved not solely by setting up an osmotic pressure butalso through the so-called colloid osmotic pressure exerted bymacromolecules. It was possible in this way to use dialysis solutionsalso in the isoosmolar or hypoosmolar range.

Employed for this purpose are, inter alia, glucose polymers which areobtained by hydrolyzing unmodified corn starch and have molecularweights of about 20 000 (icodextrin).

The residence times of such dialysis solutions are about 8 to 12 hours.Although macromolecules are also reabsorbed by means of active uptakethrough the lymphatic system, and then degraded, the degradationproducts in the form of maltose and glucose oligomers are regarded asnon-critical.

A considerable disadvantage of such hydrolyzed starch fractions is,however, regarded as being the limitation on the molecular weight by thedecrease in water solubility, which is therefore not in the optimalrange for the desired use, as the molecular weight grows. In addition,starch fragments with little or no branching are prone toretrogradation—generally known for the amylose content of starch—and maylead to unwanted precipitations. This applies all the more whenamylose-rich starches are chosen as initial basis for the hydrolyzates.In addition, maltodextrin-like starch products are prone under theconditions of autoclaving to form unwanted byproducts which are harmfulin some circumstances, such as formaldehyde and aldonic acids.

A further agent which can be employed in principle in the art forproducing a colloid osmotic pressure is hydroxyethyl starch (HES) whichis effective as oncotic medium.

HES is currently the most up-to-date and most widely used volumereplacement composition. Besides different compositions of the finishedproduct, the active substance is employed in diverse variants whichdiffer through their molecular weight and through the degree and patternof substitution.

Hydroxyethyl starches are distinguished in principle from other volumereplacement compositions such as gelatin, dextrans or artificialcolloids by being well tolerated, based on the fact that the startingmaterial used for HES is waxy corn starch, a special type of starchwhich is more than 98% composed of amylopectin which has similarity inits chemical structure with the endogenous reserve substance glycogen.The remaining approximately 2% consists of amylose with little orscarcely any branching.

Like glycogen, amylopectin is composed of glucose units which are linkedtogether in the basic structure via α-1,4 linkages and at the branchpoints via α-1,6 linkages. Amylopectin with about 5% α-1,6 linkages (atapproximately every 20th glucose unit) has, however, distinctly lessbranching than glycogen with 10 to 16% α-1,6 linkages (every 6th to 10thglucose unit). Amylopectin is insoluble in water. If it were soluble, itwould be rapidly degraded by endogenous amylases and remain ineffective.Etherification with, for example, ethylene oxide or propylene oxidemakes amylopectin water-soluble, with in addition a slowing ofdegradation by amylase—depending on the degree of substitution (degreeof etherification)—thus determining, together with the molecular weightwhich is set up, the desired duration of action when HES is used forexample as volume replacement composition.

A serious disadvantage of all known hydroxyethylated and hydropropylatedstarch types is thus regarded as being the difficulty or impossibilityof complete degradation by amylase through the more or less extensivesubstitution by hydroxyethyl and hydroxypropyl groups, respectively. Asa consequence thereof, residual fragments remain in the body and areeliminated only very slowly or are stored in various organs/tissues suchas, for example, spleen, liver and lung. This may have particularlycritical effects if the dosage is relatively high and/or long-term. Itis assumed that the known side effects such as pain in the sides oritching are attributable thereto. Investigations with HES types in themolecular weight range from 40 000 to 450 000 and degrees ofsubstitution of 0.5 and 0.7 for use in peritoneal dialysis revealed thatreabsorbed HES and fragments thereof are stored in the spleen, lung andliver, so that the use of HES as colloid osmotic agent cannot becategorized as without problems. This unwanted storage of HES isattributable to the fact that, because of the high degree ofsubstitution, complete degradation of HES by endogenous amylase is notensured.

An improvement in terms of the storage problem was provided by an HEStype with the specification 130/0.4, whose pattern of substitution hasbeen optimized by a special preparation process so that the content ofhydroxyethyl side groups which is relevant for attack by amylase wasretained but at the same time the overall degree of substitution wasdecreased.

Although it was possible thereby to reduce markedly the storage of HESresidual fragments in organs/tissues, it could not be completelysuppressed.

The invention was therefore based on the object of providing an agentwhich has the advantageous properties of the hydroxyethyl andhydroxypropyl starches known in the art but no longer has thedisadvantageous properties of storage of residual fragments in organsand tissues.

It has been found that the object can be achieved with a highlybranched, unsubstituted or low-substituted starch product, i.e. with astarch which has a significantly higher degree of branching thanamylopectin and has the degree of α-1,6 branching of glycogen, or evenexceeds this, and—if substituted—has a degree of substitution MS of onlyup to 0.3, preferably of from 0.05 to 0.3.

The term MS (molar substitution) means the average number ofhydroxyethyl or hydroxypropyl groups per anhydroglucose unit. The MS isnormally measured by determining the content of hydroxyethyl orhydroxypropyl groups in a sample and computational allocation to theanhydroglucose units present therein. The MS can also be determined bygas chromatography.

The degree of branching can be determined by a gas chromatographicmethylation analysis as mol % of the α-1,4,6-glycosidically linkedanhydroglucoses in the polymer. The degree of branching is in every casean average because the starch products of the invention are polydispersecompounds.

The glucose units in starch and glycogen and in the product of theinvention are linked via α-1,4 and α-1,6 linkages. The degree ofbranching means the proportion of α-1,4,6-linked glucose units in mol %of the totality of all anhydroglucoses.

The C₂/C₆ ratio expresses the ratio or substitution at C-2 to that atC-6.

The starch products of the invention have a degree of branching of from8% to 20%, achievable by a transglucosidation step with the aid ofbranching enzymes. The starting material which can be used for thispurpose is in principle any starch, but preferably waxy starches with ahigh proportion of amylopectin or the amylopectin fraction itself. Thedegree of branching which is necessary for the use according to theinvention of the starch products is in the range from 8% to 20%,expressed as mol % of anhydroglucoses. This means that the starchproducts which can be used for the purposes of the invention have onaverage one α-1,6 linkage, and thus a branching point, every 12.5 to 5glucose units.

Preferred starch products have a degree of branching of more than 10%and up to 20% and in particular from 11 to 18%. A higher degree ofbranching means a greater solubility of the starch products of theinvention and a greater bioavailability of these dissolved starchproducts in the body.

Particular preference is given to unmodified starch products with adegree of branching of more than 10%, in particular from 11% to 18%.

The starch products of the invention can be prepared by targetedenzymatic assembly using so-called branching or transfer enzymes, whereappropriate followed by partial derivatization of free hydroxyl groupswith hydroxyethyl or hydroxypropyl groups. Instead of this it ispossible to convert a hydroxyethylated or hydroxypropylated starch byenzymatic assembly using so-called branching or transfer enzymes into astarch product of the invention. Obtaining branched starch productsenzymatically from wheat starch with a degree of branching of up to 10%is known per se and described for example in WO-A-00/66,633. Suitablebranching or transfer enzymes and the obtaining thereof are disclosed inWO-A-00/18,893, U.S. Pat. No. 4,454,161, EP-A-418,945, JP-A-2001/294,601or U.S. Pat. No. 2002/65,410. This latter publication describesunmodified starch products with degrees of branching of more than 4% andup to 10% or higher.

The enzymatic transglycosilation can be carried out in a manner knownper se, for example by incubating waxy corn starch with the appropriateenzymes under mild conditions at pH values between 6 and 8 andtemperatures between 25 and 40° C. in aqueous solution.

As for the HES types employed clinically in the art, the averagemolecular weight (M_(w)) is—depending on the application—preferably inthe range from 10 000 to 450 000 and, where appropriate, the C₂/C₆ ratiois in the range from 4 to 20. Molecular weights in the range from 10 000to 200 000, in particular 20 000 to 40 000, are preferred for use inCAPD, and molecular weights in the range from 40 000 to 450 000 arepreferred for use as plasma expander.

The molecular weight M_(w) means for the purposes of this descriptionthe weight average molecular weight. This can be determined in a mannerknown per se by various methods, i.e. by gel permeation chromatography(GPC) or high pressure liquid chromatography (HPLC) in conjunction withlight scattering and RI detection.

The C₂/C₆ ratio preferred for substituted starches is in the range from5 to 9. The formation of unwanted byproducts such as, for example,aldonic acids and formaldehyde can be avoided by processes known to theskilled worker for reducing or oxidizing aldehyde groups at the reducingend.

The high degree of branching of the starch products of the inventionincreases the solubility in water thereof to such an extent thathydroxyethyl or hydroxypropyl substitution can be wholly orsubstantially dispensed with in order to keep the starch product insolution.

A great advantage is in particular that the average molecular weight canbe increased in a suitable manner via the permeability limit of theperitoneum. The characteristic variable which can be used in this caseis also the GPC value of the so-called bottom fraction BF90% (molecularweight at 90% of the peak area as a measure of the proportion of smallermolecule fractions). A greater UF efficiency can be achieved byappropriate raising of the molecular weight with, at the same time, adrastically reduced absorption across the peritoneal membrane. At thesame time, because of the absence of or only low substitution byhydroxyethyl or hydroxypropyl groups, high molecular weight residualfragments which are produced by degradation by endogenous amylase, andwhich can no longer be further degraded by amylase and are stored inorgans or tissues, no longer occur or now occur to only a slight extent.In addition, because of the great physiological similarity to endogenousglycogen compared with prior art HES types, considerably fewer or noside effects are to be expected. Moreover, the possibility ofretrogradation and precipitations associated therewith is avoidedbecause these have been observed only on little or unbranchedamylose-like structural constituents which are unsubstituted or onlyslightly substituted.

Starting from suitable highly branched starch it is possible byprocesses known in the art to prepare with minimal effort a colloidosmotic agent which is suitable for peritoneal dialysis and which can becombined without difficulty in a manner which is likewise known withvarious electrolytes, amino acids, lactate, acetate, bicarbonate and thelike, and with other osmotically active agents, such as, for example,glucose. It is likewise possible by suitable choice of the molecularweight to obtain a product for use as volume replacement compositionwhose volume effect is additionally favored by the spatial expansion ofa starch product highly branched in this way. It is additionallypossible to adjust the residence time in the body through the choice ofthe molecular weight distribution.

The products of the invention are distinguished by having the advantagesknown in the art for hydroxyethyl starches employed in volumereplacement but no longer having the typical disadvantages thereof. Thismakes them very interesting in particular for use in peritonealdialysis. Their advantages are particularly advantageously evident inareas of application where volume replacement compositions must beadministered intravenously in a short time in relatively large amountsor, as in the case of sudden loss of hearing for example, overrelatively long periods. This is because HES is given directly into thebloodstream in volume replacement, whereas on use in peritoneal dialysisonly fractions able to pass through the peritoneum or taken up by thelymphatic route can reach the blood. Whereas, for example, the upperlimit for one administration of HES of the specification 130/0.4 iscurrently 3 g per kg of body weight and day, larger amounts of theproducts of the invention can be administered without problems.

The products of the invention are additionally distinguished by showingthe advantages of known colloid osmotic agents for peritoneal dialysiswithout having the disadvantages of the formation of harmful byproductsor the tendency to retrogradation.

The products of the invention can be employed through modification ofthe average molecular weight both in volume replacement and inperitoneal dialysis.

The invention likewise relates to dialysis solutions comprising water,the starch products of the invention and further additions customary fordialysis solutions. Examples of the latter are electrolytes, aminoacids, lactate, acetate, bicarbonate and other osmotically active agentssuch as, for example, glucose.

The starch product of the invention is normally present in the dialysissolutions of the invention in a concentration of from 2 to 10,preferably 4 to 7.5, % by weight based on the dialysis solution.

The invention further relates to volume replacement compositions (plasmaexpanders) comprising water, the starch products of the invention andfurther additions customary for plasma expanders. An example of thelatter is sodium chloride to produce a physiologically toleratedinfusion solution.

The starch product of the invention is normally present in the plasmaexpanders of the invention in a concentration of from 2 to 12,preferably 4 to 10, % by weight based on the plasma expander.

The invention further relates to the use of the starch products of theinvention in dialysis, preferably in peritoneal dialysis.

The invention further relates to the use of the starch products of theinvention as plasma expanders.

The effectiveness of the starch product of the invention is nowexplained in detail by means of the following example.

EXAMPLE

Investigation of the tissue storage after repeated administration

A controlled study was carried out on 48 female rats. Daily infusion ofa ¹⁴C-labeled starch product of the invention (average molecular weightMw 25 500 Da; molar substitution 0.15; degree of branching 12.4 mol %)or ¹⁴C-labeled HES 130/0.4 (average molecular weight Mw 135 600 Da;molar substitution 0.41; degree of branching 6.29 mol %; in each case 1g per kg of body weight) on 24 consecutive days was followed 2, 10, 22and 46 days after the last administration by investigation of the liver,lung, spleen and kidney for tissue storage. The results are shown in thefollowing table.

As is evident from the table, a significantly lower storage of theproduct of the invention (P<0.01) compared with HES 130/0.4 was found inall the tissues investigated. These results clearly demonstrate that theproduct of the invention leads to a distinctly reduced tissue storagecompared with the comparison product.

Measured radioactivity in the investigated tissues (as % of totalactivity administered)

Starch HES Starch HES Starch HES Starch HES product 130/0.4 product130/0.4 product 130/0.4 product 130/0.4 2 days after last 10 days afterlast 22 days after last 46 days after last Tissue administrationadministration administration administration Liver 0.20 1.30 0.06 0.310.02 0.20 0.01 0.06 Spleen 0.01 0.06 0.01 0.04 0.00 0.02 0.00 0.02 Lung0.02 0.08 0.01 0.04 0.01 0.03 0.00 0.03 Kidney 0.02 0.13 0.01 0.05 0.000.01 0.00 0.01

1. A modified hydroxyethyl- or hydroxypropyl-substituted starch productfor clinical use, wherein said hydroxyethyl- orhydroxypropyl-substituted starch product has a degree of branching inthe range of from 8 to 10 mol %, a degree of substitution MS of up to0.3, an average molecular weight (M_(w)) in the range of from 10000 to450000, with the proviso that said hydroxyethyl- orhydroxypropyl-substituted starch product is not derived from anamylopectin fraction.
 2. The modified hydroxyethyl- orhydroxypropyl-substituted starch product of claim 1, wherein saidmodified hydroxyethyl- or hydroxypropyl-substituted starch product has adegree of substitution MS in the range of from 0.05 to 0.3.
 3. Themodified hydroxyethyl- or hydroxypropyl-substituted starch product ofclaim 2, wherein said modified hydroxyethyl- orhydroxypropyl-substituted starch product has a degree of substitution MSin the range of from 0.05 to 0.15.
 4. The modified hydroxyethyl- orhydroxypropyl-substituted starch product of claim 1, wherein saidmodified hydroxyethyl- or hydroxypropyl-substituted starch product hasan average molecular weight (Mw) in the range from 10,000 to 40,000. 5.The modified hydroxyethyl- or hydroxypropyl-substituted starch productof claim 1, wherein said modified hydroxyethyl- orhydroxypropyl-substituted starch product has an average molecular weight(M_(w)) in the range from 40˜000 to 450,000.
 6. The modifiedhydroxyethyl- or hydroxypropyl-substituted starch product of claim 1,wherein the C2/C6 ratio of said modified hydroxyethyl- orhydroxypropyl-substituted starch product is in the range of from 4 to20.
 7. The modified hydroxyethyl- or hydroxypropyl-substituted starchproduct of claim 6, wherein said C2/C6 ratio is in the range of from 5to
 9. 8. The modified hydroxyethyl- or hydroxypropyl-substituted starchproduct of claim 1, wherein said hydroxyethyl- orhydroxypropyl-substituted starch product is hydroxyethylated starch. 9.The modified hydroxyethyl- or hydroxypropyl-substituted starch productof claim 1, wherein the reducing ends of said modified hydroxyethyl- orhydroxypropyl-substituted starch product are inactivated by oxidation orreduction.
 10. A dialysis solution comprising water and the modifiedhydroxyethyl- or hydroxypropyl-substituted starch product of claim 1.11. A plasma expander comprising water and the modified hydroxyethyl-or, hydroxypropyl-substituted starch product of claim
 1. 12. A method ofperitoneal dialysis comprising dialyzing with a dialysis solutioncomprising the modified hydroxyethyl- or hydroxypropyl-substitutedstarch product of claim 1 as colloid osmotic agent in dialysis.
 13. Amethod for volume replacement comprising administering to a patient inneed thereof a plasma expander comprising the modified hydroxyethyl- orhydroxypropyl-substituted starch product of claim
 1. 14. A modifiedunsubstituted starch product for clinical use, wherein said modifiedunsubstituted starch product has a degree of branching in the range offrom 11 to 20 mol % and an average molecular weight (M_(w)) in the rangeof from 10,000 to 450,000, with the proviso that said unsubstitutedstarch product is not derived from an amylopectin fraction.
 15. Themodified unsubstituted starch product of claim 14, wherein said modifiedunsubstituted starch product has an average molecular weight (M_(w)) inthe range from 10,000 to 40,000.
 16. The modified unsubstituted starchproduct of claim 14, wherein said modified unsubstituted starch producthas an average molecular weight (M_(w)) in the range from 40,000 to450,000.
 17. The modified unsubstituted starch product of claim 14,wherein the reducing ends of said modified unsubstituted starch productare inactivated by oxidation or reduction.
 18. A dialysis solutioncomprising water and the modified unsubstituted starch product of claim14.
 19. A plasma expander comprising water and the modifiedunsubstituted starch product of claim
 14. 20. A method of peritonealdialysis comprising dialyzing with a dialysis solution comprising themodified unsubstituted starch product of claim 14 as colloid osmoticagent in dialysis.
 21. A method for volume replacement comprisingadministering to a patient in need thereof a plasma expander comprisingthe modified unsubstituted starch product of claim 14.